Evidential deep learning for trustworthy prediction of enzyme commission number.

The rapid growth of uncharacterized enzymes and their functional diversity urge accurate and trustworthy computational functional annotation tools. However, current state-of-the-art models lack trustworthiness on the prediction of the multilabel classification problem with thousands of classes. Here, we demonstrate that a novel evidential deep learning model (named ECPICK) makes trustworthy predictions of enzyme commission (EC) numbers with data-driven domain-relevant evidence, which results in significantly enhanced predictive power and the capability to discover potential new motif sites. ECPICK learns complex sequential patterns of amino acids and their hierarchical structures from 20 million enzyme data. ECPICK identifies significant amino acids that contribute to the prediction without multiple sequence alignment. Our intensive assessment showed not only outstanding enhancement of predictive performance on the largest databases of Uniprot, Protein Data Bank (PDB) and Kyoto Encyclopedia of Genes and Genomes (KEGG), but also a capability to discover new motif sites in microorganisms. ECPICK is a reliable EC number prediction tool to identify protein functions of an increasing number of uncharacterized enzymes.

Elucidation of bacterial trehalose-degrading trehalase and trehalose phosphorylase: physiological significance and its potential applications.

Bacteria possess diverse metabolic and genetic processes, resulting in the inability of certain bacteria to degrade trehalose. However, some bacteria do have the capability to degrade trehalose, utilizing it as a carbon source, and for defense against environmental stress. Trehalose, a disaccharide, serves as a carbon source for many bacteria, including some that are vital for pathogens. The degradation of trehalose is carried out by enzymes like trehalase (EC 3.2.1.28) and trehalose phosphorylase (EC 2.4.1.64/2.4.1.231), which are classified under the glycoside hydrolase families GH37, GH15, and GH65. Numerous studies and reports have explored the physiological functions, recombinant expression, enzymatic characteristics, and potential applications of these enzymes. However, further research is still being conducted to understand their roles in bacteria. This review aims to provide a comprehensive summary of the current understanding of trehalose degradation pathways in various bacteria, focusing on three key areas: (i) identifying different trehalose-degrading enzymes in Gram-positive and Gram-negative bacteria, (ii) elucidating the mechanisms employed by trehalose-degrading enzymes belonging to the glycoside hydrolases GH37, GH15, and GH65, and (iii) discussing the potential applications of these enzymes in different sectors. Notably, this review emphasizes the bacterial trehalose-degrading enzymes, specifically trehalases (GH37, GH15, and GH65) and trehalose phosphorylases (GH65), in both Gram-positive and Gram-negative bacteria, an aspect that has not been highlighted before.

H2O2-Driven Hydroxylation of Steroids Catalyzed by Cytochrome P450 CYP105D18: Exploration of the Substrate Access Channel.

CYP105D18 supports H2O2 as an oxygen surrogate for catalysis well and shows high H2O2 resistance capacity. We report the hydroxylation of different steroids using H2O2 as a cosubstrate. Testosterone was regiospecifically hydroxylated to 2ß-hydroxytestosterone. Based on the experimental data and molecular docking, we predicted that hydroxylation of methyl testosterone and nandrolone would occur at position 2 in the A-ring, while hydroxylation of androstenedione and adrenosterone was predicted to occur in the B-ring. Further, structure-guided rational design of the substrate access channel was performed with the mutagenesis of residues S63, R82, and F184. Among the mutants, S63A showed a marked decrease in product formation, while F184A showed a significant increase in product formation in testosterone, nandrolone, methyl testosterone, androstenedione, and adrenosterone. The catalytic efficiency (kcat/Km) toward testosterone was increased 1.36-fold in the F184A mutant over that in the wild-type enzyme. These findings might facilitate the potential use of CYP105D18 and further engineering to establish the basis of biotechnological applications. IMPORTANCE The structural modification of steroids is a challenging chemical reaction. Modifying the core ring and the side chain improves the biological activity of steroids. In particular, bacterial cytochrome P450s are used as promiscuous enzymes for the activation of nonreactive carbons of steroids. In the present work, we reported the H2O2-mediated hydroxylation of steroids by CYP105D18, which also overcomes the use of expensive cofactors. Further, exploring the substrate access channel and modifying the bulky amino acid F184A increase substrate conversion while modifying the substrate recognizing amino acid S63 markedly decreases product formation. Exploring the substrate access channel and the rational design of CYP105D18 can improve the substrate conversion, which facilitates the engineering of P450s for industrial application.

Design and Evaluation of a Carrier-Free Prodrug-Based Palmitic-DEVD-Doxorubicin Conjugate for Targeted Cancer Therapy.

In the development of new drugs, typical polymer- or macromolecule-based nanocarriers suffer from manufacturing process complexity, unwanted systematic toxicity, and low loading capacity. However, carrier-free nanomedicines have made outstanding progress in drug delivery and pharmacokinetics, demonstrating most of the advantages associated with nanoparticles when applied in targeted anticancer therapy. Here, to overcome the problems of nanocarriers and conventional cytotoxic drugs, we developed a novel, carrier-free, self-assembled prodrug consisting of a hydrophobic palmitic (16-carbon chain n-hexadecane chain) moiety and hydrophilic group (or moiety) which is included in a caspase-3-specific cleavable peptide (Asp-Glu-Val-Asp, DEVD) and a cytotoxic drug (doxorubicin, DOX). The amphiphilic conjugate, the palmitic-DEVD-DOX, has the ability to self-assemble into nanoparticles in saline without the need for any carriers or nanoformulations. Additionally, the inclusion of doxorubicin is in its prodrug form and the apoptosis-specific DEVD peptide lead to the reduced side effects of doxorubicin in normal tissue. Furthermore, the carrier-free palmitic-DEVD-DOX nanoparticles could passively accumulate in the tumor tissues of tumor-bearing mice due to an enhanced permeation and retention (EPR) effect. As a result, the palmitic-DEVD-DOX conjugate showed an enhanced therapeutic effect compared with the unmodified DEVD-DOX conjugate. Therefore, this carrier-free palmitic-DEVD-DOX prodrug has great therapeutic potential to treat solid tumors, overcoming the problems of conventional chemotherapy and nanoparticles.

Heterologous protein production using Psychrobacter sp. PAMC 21119 analyzed with a green fluorescent protein-based reporter system.

Insolubility and low expression are typical bottlenecks in the production of proteins for studying their function and structure using X-ray crystallography or nuclear magnetic resonance spectroscopy. Cold-active enzymes from polar microorganisms have unique structural features that render them unstable and thermolabile, and are responsible for decreased protein yield in heterologous expression systems. To address these challenges, we developed a heterologous protein expression system using a psychrophilic organism, Psychrobacter sp. PAMC 21119, as a protein expression host with its own promoter. We screened 11 promoters and evaluated their strength using quantitative real-time polymerase chain reaction and a reporter system harboring the SfGFP gene. The highest expression was achieved using promoters RH96_RS13655 (P21119_20930) and RH96_RS15090 (P21119_23410), regardless of the temperature used. The p20930 strain exhibited a maximum expression level 19.6-fold higher than that of its control at 20 °C and produced approximately 0.5 mg of protein per gram of dry cell weight. To our knowledge, this is the first report of a low-temperature recombinant protein expression system developed using Psychrobacter sp. that can be used to express various difficult-to-express and cold-active proteins.

Structure of fish TRAF4 and its implication in TRAF4-mediated immune cell and platelet signaling.

Due to an increasing interest in immunity and signal transduction in teleost fish, important key signaling molecules associated with the immune response, including TRAF molecules, have been recently cloned and characterized. To better understand the role of TRAF4 in fish immune signaling and compare it with the human system, our study cloned the TRAF4 gene from the Antarctic yellowbelly rockcod Notothenia coriiceps (ncTRAF4) and purified the protein. Here, we report the first crystal structure of teleost fish TRAF4. Based on biochemical characterization, our findings elucidated the mechanisms through which signaling molecules gain cold adaptivity. Additionally, we identified a platelet receptor GPIbß homolog in N. coriiceps (ncGPIbß) and found that the "RRFERLFKEARRTS" region of this homolog directly binds to ncTRAF4, indicating that ncTRAF4 also recognizes the "RLXA" motif for receptor interactions and further TARF4-mediated cellular signaling. Collectively, our findings provide novel insights into the mechanisms of TRAF4-mediated immune cell and platelet signaling in fish and the structural flexibility-mediated cold adaptiveness of signaling molecules.

Emerging Immune Checkpoint Molecules on Cancer Cells: CD24 and CD200.

Cancer immunotherapy strategies are based on the utilization of immune checkpoint inhibitors to instigate an antitumor immune response. The efficacy of immune checkpoint blockade, directed at adaptive immune checkpoints, has been demonstrated in select cancer types. However, only a limited subset of patients has exhibited definitive outcomes characterized by a sustained response after discontinuation of therapy. Recent investigations have highlighted the significance of immune checkpoint molecules that are overexpressed in cancer cells and inhibit myeloid lineage immune cells within a tumor microenvironment. These checkpoints are identified as potential targets for anticancer immune responses. Notably, the immune checkpoint molecules CD24 and CD200 have garnered attention owing to their involvement in tumor immune evasion. CD24 and CD200 are overexpressed across diverse cancer types and serve as signaling checkpoints by engaging their respective receptors, Siglec-10 and CD200 receptor, which are expressed on tumor-associated myeloid cells. In this review, we summarized and discussed the latest advancements and insights into CD24 and CD200 as emergent immune checkpoint moieties, further delving into their therapeutic potentials for cancer treatment.

Structural and Biochemical Insights into Bis(2-hydroxyethyl) Terephthalate Degrading Carboxylesterase Isolated from Psychrotrophic Bacterium Exiguobacterium antarcticum.

This study aimed to elucidate the crystal structure and biochemically characterize the carboxylesterase EaEst2, a thermotolerant biocatalyst derived from Exiguobacterium antarcticum, a psychrotrophic bacterium. Sequence and phylogenetic analyses showed that EaEst2 belongs to the Family XIII group of carboxylesterases. EaEst2 has a broad range of substrate specificities for short-chain p-nitrophenyl (pNP) esters, 1-naphthyl acetate (1-NA), and 1-naphthyl butyrate (1-NB). Its optimal pH is 7.0, losing its enzymatic activity at temperatures above 50 °C. EaEst2 showed degradation activity toward bis(2-hydroxyethyl) terephthalate (BHET), a polyethylene terephthalate degradation intermediate. We determined the crystal structure of EaEst2 at a 1.74 Å resolution in the ligand-free form to investigate BHET degradation at a molecular level. Finally, the biochemical stability and immobilization of a crosslinked enzyme aggregate (CLEA) were assessed to examine its potential for industrial application. Overall, the structural and biochemical characterization of EaEst2 demonstrates its industrial potency as a biocatalyst.

Feruloyl Esterase (LaFae) from Lactobacillus acidophilus: Structural Insights and Functional Characterization for Application in Ferulic Acid Production.

Ferulic acid and related hydroxycinnamic acids, used as antioxidants and preservatives in the food, cosmetic, pharmaceutical and biotechnology industries, are among the most abundant phenolic compounds present in plant biomass. Identification of novel compounds that can produce ferulic acid and hydroxycinnamic acids, that are safe and can be mass-produced, is critical for the sustainability of these industries. In this study, we aimed to obtain and characterize a feruloyl esterase (LaFae) from Lactobacillus acidophilus. Our results demonstrated that LaFae reacts with ethyl ferulate and can be used to effectively produce ferulic acid from wheat bran, rice bran and corn stalks. In addition, xylanase supplementation was found to enhance LaFae enzymatic hydrolysis, thereby augmenting ferulic acid production. To further investigate the active site configuration of LaFae, crystal structures of unliganded and ethyl ferulate-bound LaFae were determined at 2.3 and 2.19 Å resolutions, respectively. Structural analysis shows that a Phe34 residue, located at the active site entrance, acts as a gatekeeper residue and controls substrate binding. Mutating this Phe34 to Ala produced an approximately 1.6-fold increase in LaFae activity against p-nitrophenyl butyrate. Our results highlight the considerable application potential of LaFae to produce ferulic acid from plant biomass and agricultural by-products.

Structural basis for the substrate specificity of an S-formylglutathione hydrolase derived from Variovorax sp. PAMC 28711.

S-Formylglutathione hydrolase was originally known to catalyze the hydrolysis of S-formylglutathione to formate and glutathione. However, this enzyme has a broader esterase activity toward substrates containing thioester and ester bonds. In a previous study, we identified a new S-formylglutathione hydrolase (VaSFGH) gene in the Antarctic bacterium Variovorax sp. PAMC 28711, and recombinant VaSFGH protein was purified and characterized. Previous enzyme activity assays showed that VaSFGH has high activity, especially toward short-chain p-nitrophenyl esters (C2-C4). In this study, we determined the crystal structure of substrate-free VaSFGH at a resolution of 2.38 Å. In addition, p-nitrophenyl ester-bound VaSFGH structure models were generated by molecular docking simulations to obtain structural evidence of its substrate specificity. Comparative structural analysis of the apo-form and p-nitrophenyl ester-bound VaSFGH model structures revealed that large substrates could not bind inside the hydrophobic substrate-binding pocket because of the intrinsically static and relatively small substrate-binding pocket size of VaSFGH. This study provides useful information for further protein engineering of SFGHs for industrial use.

Freezing-enhanced oxidation of iodide by hydrogen peroxide in the presence of antifreeze proteins from the Arctic yeast Leucosporidium sp.AY30.

Ice-binding proteins (IBPs), originating from Arctic or Antarctic microorganisms, have freeze-inhibiting characteristics, allowing these organisms to survive in polar regions. Despite their significance in polar environments, the mechanism through which IBPs affect the chemical reactions in ice by controlling ice crystal formation has not yet been reported. In this study, a new mechanism for iodide (I-) activation into triiodide (I3-), which is the abundant iodine species in seawater, by using hydrogen peroxide (H2O2) in a frozen solution with IBPs was developed. A significant enhancement of I- activation into I3- was observed in the presence of Arctic-yeast-originating extracellular ice-binding glycoprotein (LeIBP) isolated from Leucosporidium sp. AY30, and a further increase in the I3- concentration was observed with the introduction of H2O2 to the frozen solution (25 times higher than in the aqueous solution after 24 h of reaction). The reaction in the ice increased with an increase in LeIBP concentration. The in-situ pH measurement in ice using cresol red (CR) revealed protons accumulated in the ice grain boundaries by LeIBP. However, the presence of LeIBP did not influence the acidity of the ice. The enhanced freeze concentration effect of H2O2 by LeIBP indicated that larger ice granules were formed in the presence of LeIBP. The results suggest that LeIBP affects the formation and morphology of ice granules, which reduces the total volume of ice boundaries throughout the ice. This leads to an increased local concentration of I- and H2O2 within the ice grain boundaries. IBP-assisted production of gaseous iodine in a frozen environment provides a previously unrecognized formation mechanism of active iodine species in the polar regions.

A computational approach to identify CRISPR-Cas loci in the complete genomes of the lichen-associated Burkholderia sp. PAMC28687 and PAMC26561.

The genus Burkholderia and its strains PAMC28687 and PAMC26561 are lichen-associated bacteria isolated from the Antarctic region. Our study is the first to provide the genome sequence of the Burkholderia sp. PAMC26561 strain. The genus Burkholderia includes bacteria that are pathogenic to plants, animals, and humans. Computational analysis of complete genomes of strains from the uncategorized Burkholderia group was performed using the NCBI databank and PATRIC (for genome sequence information) and CRISPRCasFinder (online and offline versions) software in order to predict the CRISPR loci and Cas genes. The RNAfold Webserver online software was used to predict RNA secondary structures. Our study showed that strain MSMB0852 (plasmid) possesses CRISPR-Cas system Class 2, and two lichen-associated strains, PAMC28687 (chromosome I) and PAMC26561 (chromosome I), possess CRISPR-Cas system Class 1. Additionally, only the two lichen-associated strains possess a variety of Cas genes.

Crystal Structure and Biochemical Analysis of a Cytochrome P450 CYP101D5 from Sphingomonas echinoides.

Cytochrome P450 enzymes (CYPs) are heme-containing enzymes that catalyze hydroxylation with a variety of biological molecules. Despite their diverse activity and substrates, the structures of CYPs are limited to a tertiary structure that is similar across all the enzymes. It has been presumed that CYPs overcome substrate selectivity with highly flexible loops and divergent sequences around the substrate entrance region. Here, we report the newly identified CYP101D5 from Sphingomonas echinoides. CYP101D5 catalyzes the hydroxylation of ß-ionone and flavonoids, including naringenin and apigenin, and causes the dehydrogenation of α-ionone. A structural investigation and comparison with other CYP101 families indicated that spatial constraints at the substrate-recognition site originate from the B/C loop. Furthermore, charge distribution at the substrate binding site may be important for substrate selectivity and the preference for CYP101D5.

Wistin Exerts an Anti-Inflammatory Effect via Nuclear Factor-κB and p38 Signaling Pathways in Lipopolysaccharide-Stimulated RAW264.7 Cells.

Inflammation is an immune response to cellular damage caused by various stimuli (internal or external) and is essential to human health. However, excessive inflammatory responses may be detrimental to the host. Considering that the existing drugs for the treatment of inflammatory diseases have various side effects, such as allergic reactions, stomach ulcers, and cardiovascular problems, there is a need for research on new anti-inflammatory agents with low toxicity and fewer side effects. As 4',6-dimethoxyisoflavone-7-O-ß-d-glucopyranoside (wistin) is a phytochemical that belongs to an isoflavonoid family, we investigated whether wistin could potentially serve as a novel anti-inflammatory agent. In this study, we found that wistin significantly reduced the production of nitric oxide and intracellular reactive oxygen species in lipopolysaccharide-stimulated RAW 264.7 cells. Moreover, wistin reduced the mRNA levels of pro-inflammatory enzymes (inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-2)) and cytokines (interleukin (IL)-1ß and IL-6) and significantly reduced the protein expression of pro-inflammatory enzymes (iNOS and COX-2). Furthermore, wistin reduced the activation of the nuclear factor-κB and p38 signaling pathways. Together, these results suggest that wistin is a prospective candidate for the development of anti-inflammatory drugs.

Investigating the Functional Role of the Cysteine Residue in Dehydrin from the Arctic Mouse-Ear Chickweed Cerastium arcticum.

The stress-responsive, SK5 subclass, dehydrin gene, CaDHN, has been identified from the Arctic mouse-ear chickweed Cerastium arcticum. CaDHN contains an unusual single cysteine residue (Cys143), which can form intermolecular disulfide bonds. Mutational analysis and a redox experiment confirmed that the dimerization of CaDHN was the result of an intermolecular disulfide bond between the cysteine residues. The biochemical and physiological functions of the mutant C143A were also investigated by in vitro and in vivo assays using yeast cells, where it enhanced the scavenging of reactive oxygen species (ROS) by neutralizing hydrogen peroxide. Our results show that the cysteine residue in CaDHN helps to enhance C. arcticum tolerance to abiotic stress by regulating the dimerization of the intrinsically disordered CaDHN protein, which acts as a defense mechanism against extreme polar environments.

Comparison of Fatty Acid Contents and MMP-1 Inhibitory Effects of the Two Antarctic Fish, Notothenia rossii and Champsocephalus gunnari.

Total fatty-acid (FA) contents of different organs (stomach, liver, brain, and skin) of two Antarctic fish, marbled rockcod (Notothenia rossii) and mackerel icefish (Champsocephalus gunnari), were examined using gas chromatography-mass spectrometry (GC-MS). N. rossii possessed higher contents of total omega-3, where eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), the most represented omega-3 FAs, were distributed throughout all parts of the fish. The highest level of EPA was observed in the skin and that of DHA was observed in the brain of N. rossii. C. gunnari showed organ peculiarity in that most of the omega-3 FAs were found in stomach and skin. Specifically, the highest levels of EPA and DHA were both observed in the stomach. Although N. rossii and C. gunnari both inhabit the Antarctic Southern Oceans, their characteristics in terms of the composition of fatty acids were shown to vary. The extracts were also evaluated for matrix metalloproteinase-1 (MMP-1)-inhibitory activities in UVB-induced human dermal fibroblasts, where extracts of the skin and liver of N. rossii showed the most significant inhibition upon MMP-1 production. These findings provide experimental evidence that the extracts of the Antarctic fish could be utilized as bioactive nutrients, particularly in the enhancement of skin health.

Bioactive Terphenyls Isolated from the Antarctic Lichen Stereocaulon alpinum.

Three p-terphenyls (2-4)-2-hydroxy-3,5-dimethoxy-p-terphenyl (2), 2-hydroxy-3,6-dimethoxy-p-terphenyl (3), and 2,3,5,6-tetramethoxy-p-terphenyl (4)-were isolated for the first time as natural products along with seven known compounds (1, 5-10) from the Antarctic lichen Stereocaulon alpinum. Structures of the new compounds were elucidated by comprehensive analyses of 1D and 2D NMR and HREIMS experiments. Compound 3 exhibited cytotoxicity against HCT116 cells with the IC50 value of 3.76 ± 0.03 µM and also inhibited NO production in LPS-induced RAW264.7 macrophages with the IC50 value of 22.82 ± 0.015 µM.

The ultrastructure of resurrection: Post-diapause development in an Antarctic freshwater copepod.

The copepod, Boeckella poppei, is broadly distributed in Antarctic and subantarctic maritime lakes threatened by climate change and anthropogenic chemicals. Unfortunately, comparatively little is known about freshwater zooplankton in lakes influenced by the Southern Ocean. In order to predict the impact of climate change and chemicals on freshwater species like B. poppei, it is necessary to understand the nature of their most resilient life stages. Embryos of B. poppei survive up to two centuries in a resilient dormant state, but no published studies evaluate the encapsulating wall that protects theses embryos or their development after dormancy. This study fills that knowledge gap by using microscopy to examine development and the encapsulating wall in B. poppei embryos from Antarctica. The encapsulating wall of B. poppei is comprised of three layers that appear to be conserved among crustacean zooplankton, but emergence and hatching are uniquely delayed until the nauplius is fully formed in this species. Diapause embryos in Antarctic sediments appear to be in a partially syncytial mid-gastrula stage. The number of nuclei quadruples between the end of diapause and hatching. Approximately 75% of yolk platelets are completely consumed during the same time period. However, some yolk platelets are left completely intact at the time of hatching. Preservation of complete yolk platelets suggests an all-or-none biochemical process for activating yolk consumption that is inactivated during dormancy to preserve yolk for post-dormancy development. The implications of these and additional ultrastructural features are discussed in the context of anthropogenic influence and the natural environment.

Crystal structure of a novel putative sugar isomerase from the psychrophilic bacterium Paenibacillus sp. R4.

Sugar isomerases (SIs) catalyze the reversible conversion of aldoses to ketoses. A novel putative SI gene has been identified from the genome sequence information on the psychrophilic bacterium Paenibacillus sp. R4. Here, we report the crystal structure of the putative SI from Paenibacillus sp. R4 (PbSI) at 2.98 Å resolution. It was found that the overall structure of PbSI adopts the triose-phosphate isomerase (TIM) barrel fold. PbSI was also identified to have two heterogeneous metal ions as its cofactors at the active site in the TIM barrel, one of which was confirmed as a Zn ion through X-ray anomalous scattering and inductively coupled plasma mass spectrometry analysis. Structural comparison with homologous SI proteins from mesophiles, hyperthermophiles, and a psychrophile revealed that key residues in the active site are well conserved and that dimeric PbSI is devoid of the extended C-terminal region, which tetrameric SIs commonly have. Our results provide novel structural information on the cold-adaptable SI, including information on the metal composition in the active site.

Complete genome sequencing and comparative CAZyme analysis of Rhodococcus sp. PAMC28705 and PAMC28707 provide insight into their biotechnological and phytopathogenic potential.

Study of carbohydrate-active enzymes (CAZymes) can reveal information about the lifestyle and behavior of an organism. Rhodococcus species is well known for xenobiotic metabolism; however, their carbohydrate utilization ability has been less discussed till date. This study aimed to present the CAZyme analysis of two Rhodococcus strains, PAMC28705 and PAMC28707, isolated from lichens in Antarctica, and compare them with other Rhodococcus, Mycobacterium, and Corynebacterium strains. Genome-wide computational analysis was performed using various tools. Results showed similarities in CAZymes across all the studied genera. All three genera showed potential for significant polysaccharide utilization, including starch, cellulose, and pectin referring their biotechnological potential. Keeping in mind the pathogenic strains listed across all three genera, CAZymes associated to pathogenicity were analyzed too. Cutinase enzyme, which has been associated with phytopathogenicity, was abundant in all the studied organisms. CAZyme gene cluster of Rhodococcus sp. PAMC28705 and Rhodococcus sp. PAMC28707 showed the insertion of cutinase in the cluster, further supporting their possible phytopathogenic properties.